Selective inhibition of C4-PEP carboxylases

Abstract

The present invention relates to the use of a compound, a salt or solvate thereof as C4 plant selective herbicide wherein said compound has a structure according to formula (I) wherein A is a cyclic alkyl, aryl, heterocycloalkyl, or heteroaryl group, and B is a cyclic alkyl, aryl, heterocycloalkyl, or heteroaryl group, and wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are, independently of each other H or an alkyl group, and wherein integer i is 0 or 1, preferably 1, and the bond (a) is a single or double bond, and wherein in case (a) is a double bond, n is 0 and X is O or S, and wherein in case (a) is a single bond n is 1, and X is H or an alkyl group, and wherein the bond (b) is a single or double bond, and wherein in case (b) is a double bond, m and p are 0, and wherein in case (b) is a single bond m and p are both 1, and/or according to formula (II) including tautomeric structures thereof, wherein R.sup.01 and R.sup.02 are independently of each other selected from the group consisting of H, OH, carboxylic acid, ester, alkyl, alkoxy and halogen, wherein Y.sup.1 is selected from the group consisting of (S(O)2), S(O)) and (C(O)), and wherein Y.sup.2 is O, and wherein r is 0 or 1 and wherein in case r is 0, q and s are 1, and wherein in case r is 1, q and s are 0, and wherein R.sup.01, R.sup.02, R.sup.04, R.sup.05, R.sup.06, R.sup.07, R.sup.04#, R.sup.05#, R.sup.06#, R.sup.07#, R.sup.09, R.sup.010, R.sup.011 and R.sup.012 are independently of each other selected from the group consisting of H, OH, SO3H, carboxylic acid, ester, alkyl, alkoxy and halogen, said compound being capable of binding to the malate binding site comprised by a phosphoenolpyruvate carboxylase from a C4 plant, thereby inhibiting said phosphoenolpyruvate carboxylase. ##STR00001##

Claims

1. A method of using at least one compound, a salt or solvate thereof as a C4 plant selective herbicide, comprising applying said compound, salt or solvate to a plant, wherein binding of said compound to the malate binding site of aphosphoenolpyruvate carboxylase from a C3 plant is inhibited, and wherein said compound has a structure according to formula (I) ##STR00056## wherein A is ##STR00057## wherein R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are, independently of each other, selected from the group consisting of H, OH, carboxylic acid, ester, alkyl, alkoxy and halogen, or wherein two residues in ortho position to each other form a heterocyclic ring using OCH.sub.2O or OCH.sub.2CH.sub.2O, and B is ##STR00058## wherein R.sup.11, R.sup.12, R.sup.13, R.sup.14 and R.sup.15 are, independently of each other, selected from the group consisting of H, OH, carboxylic acid, ester, alkyl, alkoxy and halogen, or wherein two residues in ortho position to each other form a heterocyclic ring using OCH.sub.2O or OCH.sub.2CH.sub.2O, and wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are, independently of each other H or an alkyl group, and wherein integer i is 0 or 1, and the bond (a) is a single or double bond, and wherein in case (a) is a double bond, n is 0 and X is O or S, and wherein in case (a) is a single bond n is 1, and X is H or an alkyl group, and wherein the bond (b) is a single or double bond, and wherein in case (b) is a double bond, m and p are 0, and wherein in case (b) is a single bond m and p are both 1, and wherein said compound being capable of binding to the malate binding site comprised by a phosphoenolpyruvate carboxylase from a C4 plant, thereby inhibiting said phosphoenolpyruvate carboxylase.

2. The method according to claim 1, wherein the compound has a structure according to formula (I) and wherein n is 0, (a) is a double bond, and X is O or S.

3. The method according to claim 2, wherein (b) is a double bond and X is O.

4. The method according to claim 2, wherein (b) is a single bond and X is O.

5. The method according to claim 1, wherein n is 1, (a) is a single bond, and X is H or alkyl.

6. The method according to claim 5, wherein (b) is a double bond.

7. The method according to claim 5, wherein b is a single bond.

8. The method of claim 1, wherein i is 1.

9. The method of claim 1, wherein the binding to the malate binding site of a phosphoenolpyruvate carboxylase from a C3 plant is inhibited by steric or electrostatic constraints of a conserved arginine in the binding site of the C3 phosphoenolpyruvate carboxylase.

10. The method of claim 9, wherein said arginine residue corresponds to Arg-884 in the C3 phosphoenolpyruvate carboxylase from Flaveria pringlei.

11. The method of claim 1, wherein at least one of R.sup.6, R.sup.7, R.sup.8, R.sup.9 or R.sup.10 is OH.

12. The method of claim 2, wherein X is O.

13. The method of claim 12, wherein the compound has the structure ##STR00059## wherein R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13, R.sup.14 and R.sup.15 are, independently of each other, selected from the group consisting of H, OH, carboxylic acid, ester, alkyl, alkoxy and halogen.

14. The method of claim 13, wherein at least one of R.sup.6, R.sup.7, R.sup.8, R.sup.9 or R.sup.10 is OH.

Description

(1) The figures show:

(2) FIG. 1: Partial alignment of PEP carboxlyases derived from various C3 and C4 plants. Indicated are the conserved arginine residues in C3 PEP carboxylases, and the conserved glycine residues in C4 PEP carboxylases. Numbering is according to Flaveria pringlei and Flaveria trinervia.

(3) FIG. 2: Selective inhibition of a C4-PEPC by butein. Activity of C4 PEPC was measured over a range of butein concentrations. The half maximal (50%) inhibitory concentration (IC50) of butein for C4 PEPC was determined from these data that were measured in triplicate for each concentration. Corresponding activity of a C3 PEPC at the IC50 concentration of butein for the C4 enzyme is also indicated in the Figure.

(4) FIG. 3: Selective inhibition of a C4-PEPC by pyrocatechol violet. Activity of C4 PEPC was measured over a range of pyrocatechol violet concentrations. The half maximal (50%) inhibitory concentration (IC50) of pyrocatechol violet for C4 PEPC was determined from these data that were measured in triplicate for each concentration. Corresponding activity of a C3 PEPC at the IC50 concentration of pyrocatechol violet for the C4 enzyme is also indicated in the Figure.

(5) FIG. 4: Selective inhibition of a C4-PEPC by 1-(3-carboxy-4-hydroxyphenyl)-2-(2,5-dihydroxyphenl(ethene). Activity of C4 PEPC was measured over a range of 1-(3-carboxy-4-hydroxyphenyl)-2-(2,5-dihydroxyphenl(ethene) concentrations. The half maximal (50%) inhibitory concentration (IC50) of 1-(3-carboxy-4-hydroxyphenyl)-2-(2,5-dihydroxyphenl(ethene) for C4 PEPC was determined from these data that were measured in triplicate for each concentration. Corresponding activity of a C3 PEPC at the IC50 concentration of 1-(3-carboxy-4-hydroxyphenyl)-2-(2,5-dihydroxyphenl(ethene) for the C4 enzyme is also indicated in the Figure.

(6) FIG. 5: Inhibition of a C4-PEPC by 2-(3,4-Dihydroxyphenyl)-6,7-dimethoxyquinoxaline (compound not according to the invention). Activity of C4 PEPC was measured over a range of 2-(3,4-Dihydroxyphenyl)-6,7-dimethoxyquinoxaline concentrations. The half maximal (50%) inhibitory concentration (IC50) of 2-(3,4-Dihydroxyphenyl)-6,7-dimethoxyquinoxaline for C4 PEPC was determined from these data that were measured in triplicate for each concentration. Corresponding activity of a C3 PEPC at the IC50 concentration of 2-(3,4-Dihydroxyphenyl)-6,7-dimethoxyquinoxaline for the C4 enzyme is also indicated in the Figure.

(7) FIG. 6: Inhibition of a C4-PEPC by catechin (compound not according to the invention). Activity of C4 PEPC was measured over a range of catechin concentrations. The half maximal (50%) inhibitory concentration (IC50) of catechin for C4 PEPC was determined from these data that were measured in triplicate for each concentration. Corresponding activity of a C3 PEPC at the IC50 concentration of catechin for the C4 enzyme is also indicated in the Figure.

(8) FIG. 7: Side by side comparison of the Allosteric Feedback Inhibitor Binding Site (Malate Binding Site) in PEPC. (A) Inhibitor-binding site of C4-PEPC (F. trinervia). (B) Inhibitor Binding Site of C3-PEPC (F. pringlei). Residues Arg641, Lys829, Arg888 and Asn964 have been identified as the malate-binding motif. In C3-PEPC, Arg884 provides an additional hydrogen bond for inhibitor binding. In C4-PEPC, this residue is replaced by glycine, which is 6.9 away from the inhibitor molecule. The bound feedback inhibitor is shown in the center of the binding site and labelled Asp1. Figures were made using LIGPLOT (Wallace et al., 1995).

(9) FIG. 8: Allosteric Feedback Inhibitor Binding Site (Malate Binding Site) of C4-type PEPCs. Residues Arg641, Lys829, Arg888 and Asn964 (Flaveria numbering) have been identified as the malate-binding motif. In C3-PEPCs (see FIG. 7 B) Arg884 provides an additional hydrogen bond for inhibitor binding. In C4-PEPCs, this residue is replaced by glycine, glutamine, serine or glutamate (see Paulus et al., 2013a and 2013b). The bound feedback inhibitor is shown in the center of the binding site and labelled Asp1. Figures were made using LIGPLOT (Wallace et al., 1995).

(10) The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

EXAMPLE 1

(11) The histidine-tagged PEP carboxylase from Flaveria pringlei or from Flaveria trinervia was heterologously expressed in E. coli and purified via immobilized metal affinity chromatography (IMAC). Crystals of the C3 (F. pringlei) and the C4 (F. trinervia) PEP carboxylase were obtained from the purified proteins by microbatch vapor diffusion. Crystals where analyzed by synchrotron radiation and PEPC structures were determined from the diffraction data by molecular replacement using XDS, coot and the CCP4 suite (Kabsch, 2010; Emsley et al. 2004; CCP4, 1994).

(12) Crystal structures of PEP carboxylase from Flaveria pringlei (PDB code: 3ZGB) or from Flaveria trinervia (PDB code: 3ZGE) were superimposed by the alignment algorithm of PyMOL (DeLano, 2002) which performs a BLAST-like BLOSUM62-weighted dynamic programming sequence alignment followed by a series of refinement cycles intended to improve the fit by eliminating pairing with high relative variability. The malate binding site in the aligned crystal structures was visually inspected for structural differences in the region of 10-15 around the bound aspartate inhibitor. From this alignment the structural difference in position 884 was identified. The C3 type PEP carboxylase of Flaveria pringlei has a voluminous arginine side chain in this position, while the C4 type PEP carboxylase of Flaveria trinervia carries a small glycine residue in the corresponding position.

(13) Potential selective inhibitors of C4 PEP carboxylase were selected from a Virtual Drug Screening (VDS) approach using the program PyRX (Wolf, 2009), standard compound libraries (ChemBank, NCI DataBase, KEGG Database), a library assembled from the Plant Metabolome Database and the high resolution crystal structures of PEP carboxylases from F. pringlei and F. trinervia.

(14) In particular, the potential selective inhibitors of C4 PEP carboxylase shown in Table 3 and Table 4 as well as in FIGS. 5 and 6 were identified:

EXAMPLE 2

Confirmation of Selective Inhibition of Selected Compounds

(15) Inhibition of purified PEP carboxylases from F. pringlei and F. trinervia by compounds selected from the VDS was monitored by a spectrophotometric coupled assay with malate dehydrogenase which reduces the oxaloacetate formed by PEP carboxylase to malate. The simultaneous oxidation of NADH is followed at 340 nm with standard optical equipment. The concentration of the inhibitors selected from the VDS was varied in the coupled assay to determine the half maximal inhibitory concentration (IC50).

(16) With butein and pyrocatchol violet, the inhibition shown in FIGS. 2 and 3 of the PEP carboxylase activity of the C3 and the C4 enzyme was obtained. With 1-(3-carboxy-4-hydroxyphenyl)-2-(2,5-dihydroxyphenl(ethene), the inhibition shown in FIG. 4 of the PEP carboxylase activity of the C3 and the C4 enzyme was obtained. Further results are given in Table 3 and Table 4. Comparative examples for compounds described earlier in PCT/EP2012/076648 are given in FIGS. 5 and 6.

EXAMPLE 3

Determination of the Binding Constant of C4-Selective Inhibitors in the Presence and in the Absence of the Natural Feedback Inhibitor Malate

(17) The dissociation constants (Kd) of PEPC from F. trinervia for putative C4-selective inhibitors (for details see Table below) will be determined by microscale thermophoresis (MST, Duhr and Braun, 2006; Jerabek-Willemsen et al., 2011) using the Monolith NT.115 (NanoTemper Technologies, Munich, Germany). Measurements will be done in the absence and in the presence of the natural feedback inhibitor malate (20 mM) in order to allow or to protect binding of the C4-selective inhibitor to the allosteric malate feedback inhibitor site.

(18) Purified protein is buffered in 50 mM potassium phosphate (pH 7.6), 150 mM NaCl, 0.05% Tween 20 and labelled with NT-547-Maleimide (NanoTemper Technologies) according to the protocol of the manufacturer. The compounds are dissolved in organic solvents like DMSO, acetone or ethanol or in aqueous buffer solutions at high concentrations.

(19) For MST measurements the stock solutions are diluted with protein buffer (50 mM potassium phosphate (pH 7.6), 150 mM NaCl, 0.05% Tween 20). The above solvent is used to prepare a 1:1 serial dilution in 16 dilution steps. 10 l of the compound dilution is mixed with 10 l protein solution and filled into capillaries. The compound concentration is highest in the first capillary and decreases in the next capillaries according to the 1:1 dilution. Data are fitted according to a cooperative binding model.

(20) TABLE-US-00005 TABLE 1 Substances to be tested in binding studies by microscale thermophoresis Butein Robtein Piceatannol trans-1-(3-Carboxy-4-hydroxyphenyl)-2-(2,5-dihydroxyphenyl)ethene Pyrocatechol Violet Gallein Cresol Red Chrome Cyanine R

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